Biomedical tests are often based on the detection of an interaction between a molecule, which is present in known amount and position (the molecular probe), and an unknown molecule to be detected or unknown molecules to be detected (the molecular target molecules). In current tests, probes are laid out in the form of a substance library on supports, the so-called microarrays or chips, so that a sample can be analyzed simultaneously at various probes in a parallel manner (see, for example, J. Lockhart, E. A. Winzeler, Genomics, gene expression and DNA arrays; Nature 2000, 405, 827-836). The probes are herein usually immobilized on a suitable matrix, as is for example described in WO 00/12575 (see, for example, U.S. Pat. No. 5,412,087, WO 98/36827), or synthetically produced (see, for example, U.S. Pat. No. 5,143,854) in a predetermined manner for the preparation of the microarrays.
A typical example for the use of microarrays in biological test methods is the detection of microorganisms in samples in biomedical diagnostics. Herein, it is taken advantage of the fact that the genes for ribosomal RNA (rRNA) are dispersed ubiquitously and have sequence portions, which are characteristic for the respective species. These species-characteristic sequences are applied onto a microarray in the form of single-stranded DNA oligonucleotides. The target DNA molecules to be examined are first isolated from the sample to be examined and are equipped with markers, for example fluorescent markers. Subsequently, the labeled target DNA molecules are incubated in a solution with the probes fixed on the microarray; nonspecifically occurring interactions are removed by means of corresponding washing steps and specific interactions are detected by means of fluorescence-optical evaluation. In this manner, it is possible to detect, for example, several microorganisms simultaneously in one sample by means of one single test. In this test method, the number of detectable microorganisms theoretically only depends on the number of the specific probes, which have been applied onto the microarray.
A variety of methods and technical systems, some of which arc also commercially available, are described for the detection of molecular interactions with the aid of microarrays or probe arrays on solid surfaces.
Classical systems for the detection of molecular interactions are based on the comparison of the fluorescence intensities of spectrally excited target molecules labeled with fluorophores. Fluorescence is the capacity of particular molecules to emit their own light when excited by light of a particular wavelength. Herein, a characteristic absorption and emission behavior ensues. In analysis, a proportional increase of the fluorescence signal is assumed as labeled molecule density on the functionalized surface increases, for example, due to increasing efficiency of the molecular interaction between target and probe molecules.
In particular, quantitative detection of fluorescence signals is performed by means of modified methods of fluorescence microscopy. Herein, the light having the absorption wavelength is separated from the light having the emission wavelength by means of filters or dichroites and the measured signal is imaged on suitable detectors, like for example two-dimensional CCD arrays, by means of optical elements like objectives and lenses. In general, analysis is performed by means of digital image processing.
Hitherto known technical solutions vary regarding their optical setup and the components used. Problems and limitations can result from the signal noise (the background), which is basically determined by effects like bleaching and quenching of the dyes used, autofluorescence of the media, assembling elements, and optical components as well as by dispersions, reflections, and secondary light sources within the optical setup.
This leads to great technical effort for the setup of highly sensitive fluorescence detectors for the qualitative and quantitative comparison of probe arrays. In particular, for screening with medium and high throughputs, specially adapted detection systems are necessary, which exhibit a certain degree of automation.
For optimizing standard epifluorescence setups for reading out molecular arrays, CCD-based detectors are known, which implement the excitation of the fluorophores in the dark field by means of incident light or transmitted light for the discrimination of optical effects like dispersion and reflections (see, for example, C. E. Hooper et al., Quantitative Photon Imaging in the Life Sciences Using Intensified CCD Cameras, Journal of Bioluminescence and Chemiluminescence (1990), p. 337-344). Herein, imaging of the arrays is performed either in exposure or by means of rasterizing using higher resolution optics. The use of multispectral light sources allows a comparatively easy access to different fluorophores by means of using different excitation filters (combinations).
Further methods for the quantitative detection of fluorescence signals are based on confocal fluorescence microscopy. Confocal scanning systems, as for example described in U.S. Pat. No. 5,304,810, are based on the selection of fluorescence signals along the optical axis by means of two pinholes.
Currently, analyses based on probe arrays are normally read out fluorescence-optically (see, for example, A. Marshall and J. Hodgson, DNA Chips: An array of possibilities, Nature Biotechnology, 16, 1998, 27-31; G. Ramsay, DNA Chips: State of the Art, Nature Biotechnology, 16, January 1998, 40-44).
A variety of in particular, confocal systems are known, which are suitable for the detection of small-scale integrated substance libraries in array format, which are installed in fluidic chambers (see, for example, U.S. Pat. Nos. 5,324,633, 6,027,880, 5,585,639, WO 00/12759).
However, the above-described methods and systems can only be adapted in a very limited way for the detection of large-scale integrated molecular arrays, which are, in particular, installed in fluidic systems, in particular due to the dispersions, reflections, and optical aberrations occurring therein. Furthermore, in such large-scale integrated arrays, great demands are made concerning the spatial resolution, which could, however, up to now technically not be implemented.
Thus, there is a need for highly integrated arrays that allow for the quantitative and/or qualitative detection of the interaction between probes and targets with comparatively low technical effort and with great precision.
The increase in selectivity and the access to alternative components motivate the establishment of alternative imaging technologies such as fluorescence polarization and time-resolved fluorescence for assays bound to solid bodies. The effect of twisting the polarization axis by means of fluorophores excited in a polarized manner is used for quantification in microtiter format. Furthermore, there are approaches to set up inexpensive systems having a high throughput (HTS systems) by means of using correspondingly modified polymer foils as polarization filters (see I. Gryczcynski et al., Polarisation sensing with visual detection, Anal. Chem. 1999, 71, 1241-1251).
More recent developments utilize the fluorescence of inorganic materials, like lanthanides (M. Kwiatowski et al., Solid-phase synthesis of chelate-labelled oligonucleotides: application in triple-color ligase-mediated gene analysis, Nucleic Acids Research, 1994, 22, 13) and quantum dots (M. P. Bruchez et. al., Semiconductor nanocrystals as fluorescent biological labels, Science 1998, 281, 2013).
Optical setups for the detection of samples labeled by means of gold beads and their visualization by means of silver amplification are described in the International Patent Application WO 00/72018.
A method for the qualitative and/or quantitative detection of targets in a sample by means of molecular interactions between probes and targets on probe arrays was provided in WO 02/02810, wherein the time-dependent behavior of precipitation formation at the array elements is detected in the form of signal intensities, i.e. dynamic measurement is performed. On the basis of a curve function describing precipitation formation as a function of time, a value quantifying the interaction between probe and target on an array element and therefore the amount of targets bound is assigned to each array element.
In many tests in biomedical diagnostics, the problem occurs that the target molecules are at first not present in an amount sufficient for detection and therefore often have to be amplified from the sample prior to the actual test procedure. Typically, the amplification of DNA molecules is performed by means of the polymerase chain reaction (PCR). For the amplification of RNA, the RNA molecules have to be converted to correspondingly complementary DNA (cDNA) via reverse transcription. This cDNA can then also be amplified by means of PCR. PCR is a standard laboratory method (like, for example, in Sambrook et al. (2001) Molecular Cloning: A laboratory manual, 3rd edition, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press).
The amplification of DNA by means of PCR is comparatively fast, allows a high sample throughput in small setup volumes by means of miniaturized methods, and is efficient in operation due to automation.
However, a characterization of nucleic acids by means of mere amplification is not possible. It is rather necessary to use analysis methods like nucleic acid sequence determinations, hybridization, and/or electrophoretic separation and isolation methods for the characterization of the PCR products subsequently to the amplification.
In general, devices and methods for the amplification of nucleic acids and their detection should be designed in such a way that as few interventions of the practitioner as possible are required. The advantages of methods allowing for multiplication of nucleic acids and their detection, and in the course of which the practitioner has to intervene only to a minimal extent, are self-evident. On the one hand, contaminations are avoided. On the other hand, the reproducibility of such methods is significantly increased, as they are accessible to automation. This may also be important with respect to the pharmaceutical approval of diagnostic methods.
At present, there are a multiplicity of methods for the amplification of nucleic acids and their detection, wherein first the target material is amplified by means of PCR amplification and subsequently the identity or the genetic state of the target sequences is determined by means of hybridization against a probe array. In general, amplification of the nucleic acid molecules or the target molecules to be detected is necessary in order to have at one's disposal amounts sufficient for a qualitative and quantitative detection within the scope of the hybridization.
Both PCR amplification of nucleic acids and their detection by hybridization are subject to several elementary problems. This applies in the same manner to methods combining PCR amplification of nucleic acids and their detection by means of hybridization.
If detectable markers, for example fluorescence labeled primers, are introduced into the nucleic acid molecules to be detected or target molecules to be detected in a method, which combines PCR amplification and detection by hybridization, a washing step is usually performed before the actual detection. Such a washing step provides for the removal of the non-converted primers, which are present in great excess compared to the amplification product, as well as of such nucleotides comprising a fluorescent label, which do not participate in the detection reaction and do not specifically hybridize with the nucleic acid probes of the microarray, respectively. In this manner, the high signal background caused by these molecules is to be reduced. However, such an additional procedure step considerably slows down the detection method. Furthermore, the detectable signal is considerably reduced also for those nucleic acids to be detected, which specifically hybridize with the nucleic acid probes of the microarray. The latter is largely based on the fact that no equilibrium between the targets bound by hybridization and targets in solution does exist anymore after the washing step. Nucleic acids, which had already hybridized with the nucleic acid probes located on the array, are detached from the binding site by washing and are therefore washed away together with the dissolved molecules. Washing or rinsing steps are typically intended to perform so that the wash or rinse liquid remains in contact with the nucleic acids for a period of time less than the average detachment time of the nucleic acids already hybridized.
Thus, there is a need for highly integrated arrays that allow for the quantitative and/or qualitative detection of the interaction between probes and targets with comparatively low technical effort and with great precision.
Furthermore, there is a need for devices which allow for the performance of PCR and analysis reaction, such as a hybridization reaction, in one reaction space.
In particular, it is a problem underlying embodiments of the present invention to provide methods and devices, respectively, by which molecular interactions between probes and targets on probe arrays can be detected in a quantitative and/or qualitative manner with great precision and high sensitivity as well as in an easy-to-do and cost-efficient manner.
Furthermore, it is a problem underlying embodiments of the present invention to provide methods and devices, respectively, for the amplification and for the qualitative and quantitative detection of nucleic acids, by which the interventions of the practitioner in the detection procedure can be minimized.
It is a further problem underlying embodiments of the present invention to provide methods and devices, respectively, for the qualitative and quantitative detection of target molecules, by which a high signal-to-noise ratio in the detection of interactions on the microarray is ensured without impairing the interaction between the target molecules and the probe molecules on the array.
It is a further problem underlying embodiments of the present invention to provide devices and methods, respectively, by which a high dynamic resolution in detection reaction is achieved, i.e. the detection of weak probe/target interactions is ensured aside of strong signals.
Furthermore, it is a problem underlying embodiments of the present invention to provide devices and methods, respectively, which allow an almost simultaneous amplification and characterization of nucleic acids at a high throughput rate.